Mr-method for the in vivo measurement of temperature or ph-value by means of a hyperpolarised contrast agent

ABSTRACT

The present invention provides a method of MR investigation of a sample, said method comprising: (i) nuclear spin polarising a high T1 MR imaging agent which contains in its molecular structure at least two hyperpolarisable nuclei within the same molecule, the frequency difference between the two resonance lines from said nuclei, δv, being dependent upon either the temperature or the pH of said sample; (ii) administering the nuclear spin polarised MR imaging agent to said sample; (iii) exposing said sample to a radiation at a frequency selected to excite nuclear spin transitions in said MR imaging agent; and (iv) detecting and manipulating magnetic resonance signals from said sample using a single-shot RARE acquisition sequence with shifted data acquisition.

[0001] This invention relates to a method of magnetic resonance imaging(MRI).

[0002] MRI is a diagnostic technique that has become particularlyattractive to physicians as it is noninvasive and does not involveexposing the patient under study to potentially harmful radiation suchas X-rays.

[0003] Techniques have been developed which involve ex vivo nuclear spinpolarisation of agents containing non zero nuclear spin nuclei (e.g.³He), prior to administration and in vivo MR signal measurement. Somesuch techniques involve the use of polarising agents, for exampleconventional OMRI contrast agents or hyperpolarised gases to achieve exvivo nuclear spin polarisation of non zero nuclear spin nuclei in anadministrable MR imaging agent. By polarising agent is meant any agentsuitable for performing ex vivo polarisation of an MR imaging agent.

[0004] MRI methods involving ex vivo nuclear spin polarisation may beimproved by using nuclear spin polarised MR imaging agents comprising intheir molecular structure nuclei capable of emitting MR signals in auniform magnetic field (e.g. MR imaging nuclei such as ¹³C or ¹⁵Nnuclei) and capable of exhibiting a long T₁ relaxation time, andpreferably additionally a long T₂ relaxation time. Such agents arereferred to hereinafter as “high T₁ agents”. A high T₁ agent, a termwhich does not include ¹H₂O, will generally be water-soluble and have aT₁ value of at least 6 seconds in D₂O at 37° C. and at a field of 7T,preferably 8 secs or more, more preferably 10 secs or more, especiallypreferably 15 secs or more, more especially preferably 30 secs or more,yet more especially preferably 70 secs or more, even yet more especiallypreferably 100 secs or more. Unless the MR imaging nucleus is thenaturally most abundant isotope, the molecules of a high T₁ agent willpreferably contain the MR imaging nucleus in an amount greater than itsnatural isotopic abundance (i.e. the agent will be “enriched” with saidnuclei).

[0005] Hyperpolarisation of an active MR nucleus may be achieved in avariety of ways, for example by polarisation transfer from a noble gas,or by one of the following methods as described in earlier publishedapplications of the present applicant; “brute force” (WO-A-99/35508),dynamic nuclear polarisation or DNP (WO-A-98/58272), parahydrogen orp-H₂ (WO-A-99/24080).

[0006] Furthermore, the use of hyperpolarised MR contrast agents in MRinvestigations such as MR imaging has the advantage over conventional MRtechniques in that the nuclear polarisation to which the MR signalstrength is proportional is essentially independent of the magneticfield strength in the MR apparatus. Currently the highest obtainablefield strengths in MR imaging apparatus are about BT, while clinical MRimaging apparatus are available with field strengths of about 0.2 to1.5T. Since superconducting magnets and complex magnet construction arerequired for large cavity high field strength magnets, these areexpensive. Using a hyperpolarised contrast agent, since the fieldstrength is less critical it is possible to make images at all fieldstrengths from earth field (40-50 μT) up to the highest achievablefields. However there are no particular advantages to using the veryhigh field strengths where noise from the patient begins to dominateover electronic noise (generally at field strengths where the resonancefrequency of the imaging nucleus is 1 to 20 MHz) and accordingly the useof hyperpolarised contrast agents opens the possibility of highperformance imaging using low cost, low field strength magnets.

[0007] It has previously been found, see for example WO-A99/35508 (toNycomed Imaging AS), that MR imaging agents (high T₁ agents) may benuclear spin polarised in the solid state prior to being dissolved in aphysiologically tolerable solvent and subsequently administered as ahyperpolarised solution to the sample under investigation. Furthermore,when the polarization is effected by means of a polarising agent, thewhole, substantially the whole, or at least a portion of the polarisingagent can be separated from the MR imaging agent prior toadministration.

[0008] When the previously disclosed MR imaging agents (high T₁ agents)are administered to a sample and said sample is exposed to a radiationat a frequency selected to excite nuclear spin transitions in selectednuclei in the agent, then by detecting the magnetic resonance signalsfrom the sample it is possible to generate an image, dynamic flow data,diffusion data, perfusion data, physiological data or metabolic datafrom said detected signals. Such physiological data can includetemperature, pH, pO₂, pCO₂ and ionic concentration, preferably pH andtemperature.

[0009] The methods previously described require the B_(o)-fieldinhomogeneity to be measured during the investigation of the sample(where B, is the primary magnetic field). It has now been found that ifan MR imaging agent (a high T₁ agent) is chosen which contains twohyperpolarised nuclei within the same molecule each with a differentsensibility to a parameter such as pH or temperature but preferably eachbeing of the same type of MR imaging nuclei, then an “internalreference” will effectively exist for the phase shift, making itunnecessary for separate field-mapping scans, used to measure theB_(o)-field inhomogeneities, to be performed.

[0010] Thus viewed from one aspect the present invention provides amethod of MR investigation of a sample, preferably of a human ornon-human animal body (e.g. a mammalian, reptilian or avian body), saidmethod comprising:

[0011] (i) nuclear spin polarising a MR imaging agent, wherein saidagent is a high T₁ agent and contains in its molecular structure atleast two hyperpolarisable nuclei of the same type of MR imaging nucleiwithin the same molecule, having similar signal amplitudes, and whereinthe frequency difference between the two resonance lines from saidnuclei, δv, is dependent upon either the temperature, pH, pO₂, pCO₂ orionic concentration of said sample;

[0012] (ii) administering the nuclear spin polarised MR imaging agent tosaid sample;

[0013] (iii) exposing said sample to a radiation at a frequency selectedto excite nuclear spin transitions in selected nuclei therein;

[0014] (iv) detecting and manipulating magnetic resonance signals fromsaid sample using a single-shot RARE acquisition sequence with shifteddata acquisition, and wherein the excitation and detection steps iii)and iv) are such that said nuclei are all being excited and detected inthe same sequence; and

[0015] (v) optionally generating an image, dynamic flow data, diffusiondata or physiological and/or metabolic data from said detected signals.

[0016] Thus the invention involves the sequential steps of nuclear spinpolarizing (otherwise referred to herein as “hyperpolarising”) an MRimaging agent containing in its molecular structure at least twohyperpolarisable nuclei of the same type of MR imaging nuclei within thesame molecule, having similar signal amplitudes, and wherein thefrequency difference between the two resonance lines from said nuclei,δv, is dependent upon either the temperature, pH, pO₂, pCO₂ or ionicconcentration of said sample, administering the nuclear spin polarisedMR imaging agent to a sample (preferably in solution but optionally as afinely divided particulate, and preferably in the absence of a portionof, more preferably substantially the whole of, the species involved intransferring the polarisation), preferably by bolus injection, andperforming in vivo MR signal generation and measurement using asingle-shot RARE-acquisition sequence with shifted data acquisition. TheMR signals obtained in this way may be conveniently converted byconventional manipulations into 2-, 3- or 4-dimensional data includingflow, diffusion, physiological or metabolic data.

[0017] By “hyperpolarised” we mean polarised to a level over that foundat room temperature and 1T, preferably polarised to a polarisationdegree in excess of 0.1%, more preferably 1%, even more preferably 10%.

[0018] By “physiologically tolerable solvent” we mean any solvent,solvent mixture or solution that is tolerated by the human or non-humananimal body, e.g. water, aqueous solutions such as saline,perfluorocarbons, etc.

[0019] Preferably, the frequency difference noted in step (i) of themethod of the present invention is greater than 0.5 Hz per K or per 0.1pH unit, more preferably greater than 1 Hz per K or per 0.1 pH unit, yetmore preferably greater than 2 Hz per K or per 0.1 pH unit, even morepreferably greater than 5 Hz per K or per 0.1 pH unit, most preferablybetween 10 Hz and 50 Hz per K or per 0.1 pH unit.

[0020] The frequency difference is primarily sensitive to eithertemperature or pH, although small interferences between the two cannever be totally avoided.

[0021] According to the present invention, polarisation may be achievedby use of a polarising agent. The species providing the nuclear spinpolarisation to the MR imaging agent is preferably separated asthoroughly as possible from the MR imaging agent once the transfer ofpolarisation has taken place. Preferably at least 80% of thepolarisation transferring material is removed, particularly preferably90% or more, especially preferably 95% or more, most especially 99% ormore.

[0022] In the separation step of the method of the invention, it isdesirable to remove substantially the whole of the polarisation transferagent from the composition (or at least to reduce it to physiologicallytolerable levels) as rapidly as possible. Many physical and chemicalseparation or extraction techniques are known in the art and may beemployed to effect rapid and efficient separation of the polarizationtransfer agent and MR imaging agent.

[0023] As noted above, the method according to the present invention canbe used to provide physiological data (pH, pO₂, pCO₂, temperature orionic concentrations), preferably pH and temperature data. Although suchpreferred data can be obtained via other more conventional methods, suchother methods suffer from several drawbacks. Possible other methods (andtheir drawbacks compared to the method of the present invention)include:

[0024] variation of M_(o)-magnetisation, T₁-relaxation times orT₂-relaxation times (low sensitivity/measurement precision also low);

[0025] variation of diffusion coefficient (indirect method/diffusioncoefficient may vary for reasons other than temperaturechanges/sensitive to sample motion);

[0026] localised spectroscopy measuring chemical shift (poorresolution/not suitable for temperature/pH mapping);

[0027] chemical shift imaging (poor resolution/time consuming dataacquisition)

[0028] Earlier patents in this area mention the possibility ofestimating temperatures by NMR (seer for example, U.S. Pat. No.4,558,279, U.S. Pat. No. 5,207,222, U.S. Pat. No. 5,327,884, U.S. Pat.No. 5,378,987, U.S. Pat. No. 5,690,909 and WO-A-97/20193), estimating pHvalues by NMR (see, for example, U.S. Pat. No. 5,210,290 and U.S. Pat.No. 5,639,906) or both (see, for example, U.S. Pat. No. 0,095,124).However these earlier patents do not mention techniques as hereinclaimed where hyperpolarised MR imaging agents containing twohyperpolarisable nuclei are administered to a sample, followed by invivo MR signal generation and measurement using a single-shotRARE-acquisition sequence.

[0029] Step (iv) of the method according to the present inventioncomprises a spin-echo sequence. For instance, the NMR-signal may arisefrom a substance showing two resonance lines, separated by a frequencydifference δv (see FIG. 1 of the accompanying drawings). When theresonance lines are of equal intensity, the relative phase shift of thelines can create either signal amplification or complete signalcancellation. This technique is known in clinical MRI as the“in-phase/out-of-phase” technique and is used in conjunction withgradient echo imaging, where the echo time (TE) controls the amount ofdephasing.

[0030] The level of image contrast with the “in-phase/out-of-phase”technique depends on the relative phase between the two spin populationsand not the absolute phase. Therefore there is no requirement to correctthe images for B_(o)-inhomogeneities and/or motion of the sample. Afurther advantage of this technique is that the contrast is obtained viaa single image and the method of the present invention is therefore notlinked to cases of measuring a temperature change between two images.

[0031] Although, as stated above, there is no requirement to correct forB_(o)-inhomogeneities, gradient echo imaging in general suffers from asensitivity to susceptibility variations, for example, in the abdomen ornear the lungs. Such problems are particularly acute when using longecho times, as is necessary when separating NMR-lines with a smalldifference in frequency or small chemical shift, i.e. δv is small. Ithas now been found that by using a spin echo sequence comprising arefocusing 180°-pulse alleviates the problems of sensitivity tosusceptibility variations. Conventionally, a spin echo sequence couldnot be used for detecting the phase difference of two spin populationssince the 180°-pulse rephases the spins and cancels any chemical shifteffect. In such cases the data acquisition coincides with the spin echo.However, should the read-out gradient and data acquisition be displacedby a time τ relative to the spin echo, then the spin echo sequence canstill be used to detect the relative phase difference.

[0032] A single-shot RARE-sequence where only a single 90° RF-pulse isused is utilised in a method according to the present invention (seeFIG. 3 of the accompanying drawings). Indeed, when hyperpolarisedsubstances are used, it is not possible to apply more than one 90°-pulsedue to the fact that there is no recovery of longitudinal magnetisation.Therefore, the single-shot RARE sequence is fully compatible with theuse of hyperpolarised substances in methods according to the presentinvention.

[0033] The single-shot RARE sequence, which is based on a standardRARE-sequence, will now be described in greater detail, the letterscorresponding to those on FIG. 3 of the accompanying drawings.

[0034] A) Shifted Data Acquisition

[0035] The shift is given by parameter τ. This shift is a prerequisitefor phase sensitive imaging.

[0036] B) Single-Shot RARE-Acquisition

[0037] The single-shot technique enables the imaging of substances withvery long T₁ times to be performed, e.g. ¹³C-imaging. The spin-echotechnique described herein suppresses susceptibility artefacts.

[0038] C) Extra 180°-Pulse Without Data Acquisition

[0039] An extra 180°-pulse suppresses artefacts arising from stimulatedechoes in combination with centred-phase encoding (see step D).

[0040] D) Centred-Phase Encoding

[0041] Minimises the influence of the gradual loss of phase coherencedue to T₂-relaxation at long echo times.

[0042] E) Spin Preparation

[0043] Proton decoupling used together with, e.g. ¹³C imaging, may givesome improvements (due to sharper lines).

[0044] F) Flip Back

[0045] Restores part of the longitudinal magnetisation after a scan.This is particularly useful when the T₁-relaxation time is long andhence averaging over several scans is performed in order to increase thesignal-to-noise ratio.

[0046] The novel modification in this sequence is the shifted dataacquisition stage (A), which enables phase-sensitive signal detection.In standard RARE- or SE-sequences, the data acquisition is not shifted,i.e. data acquisition coincides with the spin echo. The sequencedescribed above therefore forms a further aspect of the presentinvention.

[0047] Although the choice of a single-shot RARE sequence makes possiblethe imaging of hyperpolarised substances, the imaging principle itselfdoes not itself require hyperpolarised nuclei to be present, andtherefore the sequence forming a further aspect of the invention can beused to generate physiological data, particularly pH and temperaturedata, from any non-zero nuclei spin nuclei (e.g. ¹H, ³Li, ¹³C ¹⁵N ¹⁹F,²⁹Si or ³¹P)

[0048] Suitable MR imaging agents for use in the method of the presentinvention should satisfy the following criteria:

[0049] the agent should possess two resonance lines separated by afrequence difference δv;

[0050] this frequency difference, δv, should preferably be dependent oneither the temperature or pH of the sample;

[0051] the signal amplitude from each of the two resonance lines shouldbe similar, preferably equal; and

[0052] the agent should exhibit a long T₂ relaxation time, preferablygreater than 0.5 sees, more preferably greater than 1 sec, even morepreferably than 5 secs.

[0053] Suitable MR imaging agents, high T₁ agents, may contain nucleisuch as protons. However other non-zero nuclear spin nuclei may beuseful (e.g. ¹⁹F, ³Li, ¹³C, ¹⁵N, ²⁹Si or ³¹P, as well as ¹H), preferably¹H, ¹³C, ¹⁵N, ¹⁹F, ²⁹Si and ³¹P nuclei, with ¹³C, ¹⁵N, ¹⁹F and ³¹Pnuclei being particularly preferred. In this event the MR signals fromwhich the image is generated may be substantially only from the MRimaging agent itself.

[0054] As noted above, ¹H, ¹³C, ¹⁹N, ¹⁹F and ³¹P are the nuclei mostsuited to use in a method of the present invention. ¹H nuclei have theadvantages of being present in high concentration in natural abundanceand having the highest sensitivity of all nuclei. ¹³C nuclei areadvantageous as almost all the signal from such nuclei will be from thehyperpolarised resonance lines that are useful for generatingphysiological data, particularly temperature and pH data. The backgroundsignal from hyperpolarised ¹³C nuclei is low and much less than from,for example, ¹H nuclei. ¹⁹F nuclei have high sensitivity (88% of thatfrom ¹H nuclei, for instance), a gyromagnetic ratio 94% that from ¹H andalso gives no background signal.

[0055] Where the MR imaging nucleus is other than a proton (e.g. ¹³C,¹⁹F or ¹⁵N), there will be essentially no interference from backgroundsignals (the natural abundance of ¹³C and ¹⁵N, for instance, beingnegligible) and image contrast will be advantageously high. This isespecially true where the MR imaging agent itself is enriched abovenatural abundance in the MR imaging nucleus. Thus the method accordingto the invention has the benefit of being able to provide significantspatial weighting to a generated image.

[0056] In one embodiment, a “native image” of the sample (e.g. body)(i.e. one obtained prior to administration of the MR imaging agent orone obtained for the administered MR imaging agent without priorpolarisation as in a conventional MR experiment) may be generated toprovide structural (e.g. anatomical) information upon which the imageobtained in the method according to the invention may be superimposed. A“native image” is generally not available where ¹³C or ¹⁵N is theimaging nucleus because of the low abundance of ¹³C and ¹⁵N in the body.In this case, a proton MR image may be taken to provide the anatomicalinformation upon which the ¹³C or ¹⁵N image may be superimposed.

[0057] The MR imaging agent should of course be physiologicallytolerable or be capable of being provided in a physiologicallytolerable, administrable form where the sample is animate. Preferred MRimaging agents are soluble in aqueous media (e.g. water) and are ofcourse non-toxic where the intended end use is in vivo.

[0058] Conveniently, the MR imaging agent once polarised will remain sofor a period sufficiently long to allow the imaging procedure to becarried out in a comfortable time span. Generally sufficientpolarisation will be retained by the MR imaging agent in itsadministrable form (e.g. in injection solution) if it has a T₁ value (ata field strength of 0.01-5T and a temperature in the range 20-40° C.) ofat least 5 s, more preferably at least 10 s, especially preferably 30 sor longer, more especially preferably 70 s or more, yet more especiallypreferably 100 s or more (for example at 37° C. in water at 1T and aconcentration of at least 1 mM). The MR imaging agent may beadvantageously an agent with a long T₂ relaxation time.

[0059] Alternatively, the T₂ valve may be sensitive to the physiologicalparameters of interest.

[0060] Solid MR imaging agents (e.g. ¹³C or ¹⁵N enriched solids) mayexhibit very long T₁ relaxation times and for this reason are especiallypreferred for use in the present method.

[0061] For in vivo use, a polarised solid MR imaging agent is dissolvedin administrable media (e.g. water or saline), administered to a subjectand conventional MR imaging performed. Thus solid MR imaging agents arepreferably rapidly soluble (e.g. water soluble) to assist in formulatingadministrable media. Preferably the MR imaging agent should dissolve ina physiologically tolerable carrier (e.g. water or Ringers solution) toa concentration of at least 1 mM at a rate of 1 mM/3T, or more,particularly preferably 1 mM/2T₁ or more, especially preferably 1 mM/T₁or more. Where the solid MR imaging agent is frozen, the administrablemedium may be heated, preferably to an extent such that the temperatureof the medium after mixing is close to 37° C.

[0062] A polarised MR imaging agent may be administered (either alone orwith additional components such as additional MR imaging agents) inliquid form. The retention of polarisation in a liquid medium vis-a-visa gas medium is significantly greater. Thus while T₁ and T₂ are ingeneral shorter for the liquid, the T₂* effect due to diffusion is 10⁵times less significant for the liquid.

[0063] The MR imaging agent should be preferably enriched with nuclei(e.g. ¹⁵N and/or ¹³C nuclei) having a long TL relaxation time. Preferredare ¹³C enriched MR imaging agents having ¹³C at two particularpositions (to about the same level of enrichment) in an amount in excessof the natural abundance, i.e. above about it. Preferably these carbonpositions will have 51 or more ¹³C, particularly preferably 10% or more,especially preferably 25% or more, more especially preferably 50% ormore, even more preferably in excess of 99% (e.g. 99.9%). The ¹³C nucleishould preferably amount to >2% of all carbon atoms in the compound. TheMR imaging agent is preferably ¹³C enriched at carbonyl or quaternarycarbon positions, given that a ¹³C nucleus in a carbonyl group or incertain quaternary carbons may have a T₁ relaxation time typically ofmore than 28, preferably more than 5 s, especially preferably more than30 s. Preferably the ¹³C enriched compound should be deuterium labelled,especially adjacent the ¹³C nucleus.

[0064] Preferred ¹³C enriched compounds are those in which the ¹³Cnuclei are surrounded by one or more non-MR active nuclei such as O, S,C or a double bond.

[0065] Viewed from a further aspect, the present invention provides amethod of MR investigation of a sample previously administered with anuclear spin polarised MR imaging agent formed by nuclear spinpolarising a MR imaging agent, wherein said agent is a high T₁ agent andcontains in its molecular structure at least two hyperpolarisable nucleiof the same type of MR imaging nuclei within the same molecule havingsimilar signal amplitudes, and wherein the frequency difference betweenthe two resonance lines from said nuclei, δv, is dependent upon eitherthe temperature, pH, pO₂, pCO₂ or ionic concentration of said sample,said method comprising:

[0066] i) exposing said sample to a radiation at a frequency selected toexcite nuclear spin transitions in selected nuclei therein;

[0067] (ii) detecting and manipulating magnetic resonance signals fromsaid sample using a single-shot RARE acquisition sequence with shifteddata acquisition, and wherein the excitation and detection steps iii)and iv) are such that said nuclei are all being excited and detected inthe same sequence; and

[0068] (iii) optionally generating an image, dynamic flow data,diffusion data or physiological and/or metabolic data from said detectedsignals.

[0069] It is envisaged that, in the method according to the invention,the level of polarisation achieved should be sufficient to allow thehyperpolarised solution of the MR imaging agent to achieve adiagnostically effective contrast enhancement in the sample to which itis subsequently administered in whatever form. In general, it isdesirable to achieve a degree of polarization which is at least a factorof 2 or more above the equilibrium value at the temperature and themagnetic field in which MRI is performed, preferably a factor of 10 ormore, particularly preferably 100 or more and especially preferably 1000or more, e.g 50000.

[0070] The MR imaging agents used in the method according to theinvention may be conveniently formulated with conventionalpharmaceutical or veterinary carriers or excipient B.

[0071] For use in in vivo imaging, the formulation, which preferablywill be substantially isotonic, may conveniently be administered at aconcentration sufficient to yield a 1 micromolar to 10M concentration ofthe MR imaging agent in the imaging zone; however the preciseconcentration and dosage will of course depend upon a range of factorssuch as toxicity, the organ targeting ability of the MR imaging agentand the administration route.

[0072] Parenterally administrable forms should of course be sterile andfree from physiologically unacceptable agents, and should have lowosmolality to minimize irritation or other adverse effects uponadministration and thus the formulation should preferably be isotonic orslightly hypertonic.

[0073] Where the MR imaging agent is to be injected, it may beconvenient to inject simultaneously at a series of administration sitessuch that a greater proportion of the vascular tree may be visualizedbefore the polarization is lost through relaxation.

[0074] The dosages of the MR imaging agent used according to the methodof the present invention will vary according to the precise nature ofthe MR imaging agents used, of the tissue or organ of interest and ofthe measuring apparatus. Preferably the dosage should be kept as low aspossible while still achieving a detectable contrast effect. In general,the maximum dosage will depend on toxicity constraints.

[0075] The contents of all publications referred to herein are herebyincorporated by reference.

[0076] The invention is illustrated with reference to the followingnon-limiting Examples and the accompanying drawings in which:

[0077]FIG. 1 shows the NMR signal from a substance showing two resonancelines, separated by a frequency difference δv;

[0078]FIG. 2: [NOW DELETED]

[0079]FIG. 3 shows a single-shot RARE-sequence for use in a methodaccording to the present invention;

[0080]FIG. 4 shows some examples of phantom imaging on mixtures of waterand acetic acid, using the RARE-sequence described in FIG. 3; and

[0081]FIG. 5 shows examples of phantom images from cooled samples overtime.

EXAMPLE 1

[0082] Phantom images were obtained from mixtures of water and aceticacid, using the RARE-sequence shown in FIG. 3. The images obtained areshown in FIG. 4. The top left images in FIG. 4 were produced using 16averages and τ=10 ms. The signal-to-noise ratio was 260 for a 1 minutescan time. All tubes have the same contrast. The top right images inFIG. 4 were produced using 16 averages and τ=1 ms. The signal-to-noiseratio was 240 for a 1 minute scan time. The darkest tube contains 50%water:50% acetic acid and the signal from this tube was almostcompletely cancelled. The bottom left images in FIG. 4 were producedusing a single-shot image (scan time 0.6 secs) and τ=0 ms. Thesignal-to-noise ratio was 80. All tubes have the same contrast. Thebottom right images in FIG. 4 were produced using a single-shot image(scan time 0.6 secs) and τ=1 ms. The signal-to-noise ratio was 85 for a1 minute scan time. The darkest tube contains 50% water:50% acetic acid.

EXAMPLE 2

[0083] Two identical phantoms containing a mixture of 50% water:50%acetone were prepared. One phantom was placed inside the magnet at aconstant temperature of 17° C., whilst the other was cooled to 7° C. Thecooled phantom was then placed inside the magnet and imaging of the twophantoms was started 2 minutes after the second phantom was taken out ofthe cooler. FIG. 5A shows the cooled phantom to the left, with lowintensity. FIGS. 5B-F were all acquired at consecutive 5 minuteintervals, i.e. 7, 12, 17, 22 and 27 minutes after removal from thecooler, respectively. The right-hand phantom at constant temperatureremains bright with a constant intensity throughout all images, whereasthe intensity of the cooled phantom gradually increases as it getswarmer with time.

1. A method of MR investigation of a sample, said method comprising: (i)nuclear spin polarising a MR imaging agent, wherein said agent is a highT₁ agent and contains in its molecular structure at least twohyperpolarisable nuclei of the same type of MR imaging nuclei within thesame molecule, having similar signal amplitudes, and wherein thefrequency difference between the two resonance lines from said nuclei,by, is dependent upon either the temperature, pH, pO₂, pCO₂ or ionicconcentration of said sample; (ii) administering the nuclear spinpolarised MR imaging agent to said sample; (iii) exposing said sample toa radiation at a frequency selected to excite nuclear spin transitionsin selected nuclei therein; (iv) detecting and manipulating magneticresonance signals from said sample using a single-shot RARE acquisitionsequence with shifted data acquisition, and wherein the excitation anddetection steps iii) and iv) are such that said nuclei are all beingexcited and detected in the same sequence; and (v) optionally generatingan image, dynamic flow data, diffusion data or physiological and/ormetabolic data from said detected signals.
 2. A method as claimed inclaim 1 wherein said hyperpolarisable nuclei in said high T₁ agent aretwo non-hydrogen spin ½ nuclei.
 3. A method as claimed in claim 2wherein said nuclei are two ¹³C nuclei.
 4. A method as claimed in claim1 wherein said administration in step ii) is by bolus injection.
 5. Amethod as claimed in claim 1 wherein said administration in step ii) isafter said agent has undergone dissolution in a physiologicallytolerable solvent.
 6. A method as claimed in claim 1 wherein saidadministration in step ii) is after said agent is separated from some orall of the species providing the nuclear spin polarisation to saidagent.
 7. A method as claimed in claim 6 wherein at least 8Bo of thepolarisation transferring material is removed.
 8. A method as claimed inclaim 1 wherein said radiation in step iii) is at a frequency selectedto excite nuclear spin transitions in the spin polarised nuclei of saidMR imaging agent.
 9. A method as claimed in any one of the precedingclaims wherein said high T₁ agent is water soluble.
 10. A method asclaimed in any one of the preceding claims wherein said high T₁ agenthas a T₁ value of at least 6 secs in D₂O at 37° C. and at a field of 7T.11. A method as claimed in claim 10 wherein said T₁ value is 100 secs ormore.
 12. A method as claimed in any one of the preceding claims whereinsaid high T₁ agent contains said hyperpolarisable nuclei in an amountgreater than isotopic abundance.
 13. A method as claimed in claim 1wherein δv is dependent upon either the temperature or pH of the sample.14. A method as claimed in claim 13 wherein said frequency difference instep i) is greater than 0.5 Hz per K or per 0.1 pH unit.
 15. A method asclaimed in claim 13 wherein said frequency difference in step i) isbetween 10 Hz and 50 Hz per K or per 0.1 pH unit.
 16. A method asclaimed in any one of the preceding claims wherein said MR imaging agentcomprises ¹³C nuclei at two particular positions in an amount in excessof natural abundance and wherein the ¹³C nuclei amounts to >2% of allcarbon atoms in the agent.
 17. A method as claimed in claim 16 whereinsaid ¹³C nuclei are present in said agent at said positions to a levelof enrichment in excess of 99%.
 18. A method as claimed in either claim16 or claim 17 wherein said agent is ¹³C enriched at carbonyl orquaternary carbon positions.
 19. A method as claimed in any one ofclaims 16 to 18 wherein said ¹³C nuclei are surrounded by one or morenon-MR active nuclei.
 20. A method of MR investigation of a samplepreviously administered with a nuclear spin polarised MR imaging agentformed by nuclear spin polarising a MR imaging agent, wherein said agentis a high T₁ agent and contains in its molecular structure at least twohyperpolarisable nuclei of the same type of MR imaging nuclei within thesame molecule having similar signal amplitudes, and wherein thefrequency difference between the two resonance lines from said nuclei,δv, is dependent upon either the temperature, pH, pO₂, pCO₂ or ionicconcentration of said sample, said method comprising: i) exposing saidsample to a radiation at a frequency selected to excite nuclear spintransitions in selected nuclei therein; (ii) detecting and manipulatingmagnetic resonance signals from said sample using a single-shot RAREacquisition sequence with shifted data acquisition, and wherein theexcitation and detection steps iii) and iv) are such that said nucleiare all being excited and detected in the same sequence; and (iii)optionally generating an image, dynamic flow data, diffusion data orphysiological and/or metabolic data from said detected signals.
 21. Amethod as claimed in any one of the preceding claims wherein the said atleast two hyperpolarisable nuclei of the same type of MR imaging nucleiwithin the same molecule have the same signal amplitude.